The present invention relates to a method for reversibly inactivating enzymes and, in particular, for reversibly inactivating DNA polymerases and ligases.
The DNA polymerase isolated from Thermus aquaticus (Taq) is widely used in the polymerase chain reaction (PCR) to amplify small amounts of DNA and RNA (with reverse transcriptase in RT-PCR) by many orders of magnitude. Being a thermostable protein, it is resistant although not totally unaffected by the heat denaturation required to create single-stranded DNA from double-stranded DNA.
The PCR process itself requires four distinct phases. First, an initial DNA denaturation step, generally between 92xc2x0-96xc2x0 C. for 2-4 minutes. This is followed by another, short denaturation step (10 seconds at 92xc2x0-96xc2x0 C.), after which the primers, being short segments of DNA that are chemically synthesised to anneal very specifically to a complementary stretch of single-stranded (denatured) DNA, are allowed to anneal to the denatured DNA. The final stage is the extension step, which occurs at 72xc2x0 C. for a length of time dependent on the length of the DNA strand that needs to be synthesised. The latter 3 stages are cycled 20-30 times. Hence, each cycle of the latter three stages produces twice as many of the desired DNA fragments, resulting in an exponential increase (2n, where n=the number of cycles) in PCR product.
All four of the described stages rely on accurate temperature control in order to be accomplished properly. A variety of thermocyclers are commercially available to achieve this. Clearly, temperature control is imperative in the denaturation stages, as too low a temperature will not generate a sufficient amount of the required single stranded DNA template, whereas too high a temperature will destroy enzyme activity, which is rapidly inactivated above 94xc2x0-95xc2x0 C. Similarly, if the temperature is too low during the annealing step, the primer will bind non-specifically to the DNA, resulting in the exponential amplification of non-specific products. Too high a temperature will not allow primer-template annealing at all, and hence no product formation. Finally, the extension step at 72xc2x0 C. is an enzymatic optimum, allowing the maximum amount of product to be synthesised. Clearly, variations from that optimum will reduce PCR product yields.
Although sophisticated computer packages are available to aid in primer design, and a high level of thermal control is available on commercial thermocyclers, the problem of non-specific primer annealing persists. The principal reason for this remains the fact that when the reactants are mixed together the temperature is sub-optimal, encouraging primer-template annealing. During the subsequent elapsed time before the first denaturation temperature is reached, a small amount of non-specific annealing and extension takes place, ultimately resulting in contaminating non-specific product formation.
Currently, a number of laborious, expensive and time-consuming approaches are available to alleviate this problem. These xe2x80x9cHot Startxe2x80x9d methods include physically separating reactants until annealing temperatures are reached, either manually or by using wax, see In Innes, M. A., Gelfand, H. D., Sninsky, J. J. and White T. J. (Ed.), PCR Protocols, a Guide to Methods and Applications. Academic Press, California, USA. These not only introduce a lot of extra time into the experimental process but can introduce contaminants, due to the wax barrier itself or the requirement of opening the reaction vessel once some of the reactants have already been mixed and heated.
As discussed in Kellog, D. E. et al. (1994) Biotechniques 16,1134-1137, an antibody, specific for the active site of the enzyme is available (binding and inhibiting activity at low temperatures but becoming denatured at high temperatures), but has proved to be expensive as well as being unable to create a graduated activation response, since all the antibody will be denatured at once.
A reversibly inactivated chemically-modified version of the enzyme is available, as described and illustrated in U.S. Pat. No. 5,677,152. The contents of that prior patent are incorporated herein in their entirety by reference.
The chemically modified enzyme of that US patent is synthesised using a single phase water-based system in which both the enzyme and reagent, dicarboxylic acid anhydride, are dissolved. However, the method of preparing the modified enzyme has very strict pH, temperature and reagent excess constraints, principally because the dicarboxylic acid anhydride modifier reagent spontaneously hydrolyses in water (to form an acid) under the circumstances in question. Too much anhydride will result in a huge increase in (exothermic) acid formation, a dramatic pH drop and temperature increase, and subsequent enzyme denaturation. Too little and the vast majority of the anhydride will hydrolyse spontaneously and not remain to modify the protein!
The temperature that the reaction can be carried out at is also necessarily very limited, quoted to be below about 25xc2x0 C. but usually carried out at no higher than 4xc2x0 C. for a period as long as 12 hours (or overnight), as any increase in temperature correspondingly increases the rate of modifier reagent hydrolysis in the water, compounding the pH and enzyme denaturation problem even more. Finally, once successfully completed, the enzyme preparation is contaminated with acid. High temperatures are used to re-activate the enzyme, restoring enzyme activity.
It is a general objective of the present invention to provide a method by which a thermostable enzyme, used to amplify nucleic acids with a large reduction in non-specific product formation, can be synthesised in an inactive form so as to be subsequently activated by high temperatures, where the highlighted major problems illustrated above can be avoided.
According to a first apect of the present invention there is provided a method for reversibly inactivating thermostable DNA polymerase or ligase, which method comprises reacting a mixture of the thermostable DNA polymerase or ligase with a dicarboxylic acid anhydride, wherein the reaction is carried out using a dried DNA polymerase or ligase in an anhydrous aprotic organic solvent, the dicarboxylic acid anhydride being also substantially anhydrous, whereby the reaction results in essentially complete inactivation of enzyme activity.
Preferably the dried DNA polymerase or ligase is first suspended in the aprotic organic solvent and then to this the substantially anhydrous dicarboxylic acid anhydride is added for the reaction to take place. The reaction is suitably carried out at a temperature greater than about 30xc2x0 C.
Preferably the method comprises the further step of separating the solid phase comprising the revesibly inactivated enzyme from the liquid phase comprising the aprotic organic solvent and washing the solid phase with organic solvent.
Suitably following washing, the reversibly inactivated enzyme is dried.
The anhydrous aprotic organic solvent is preferably selected from the group comprising t-methyl butyl ether (t-MBE), butyl ether, carbon tetrachloride, cyclohexanone, ethyl acetate, methyl ethyl ketone, methyl pentanone, propyl ether, pyridine and sulfolane.
According to a second aspect of the present invention there is provided a reversibly inactivated DNA polymerase or ligase prepared by the method above.
According to a third aspect of the present invention there is provided a kit for carrying out a polymerase chain reaction comprising a reversibly inactivated DNA polymerase as defined above.